We wish to continue our efforts to determine the composition and structure of EIM, in particular the dissociation of the aggregate to its subunits and reassociation with recovery of activity. New methods are developed for the reassociation and insertion of EIM into planar bilayers. Our recent work indicates that EIM is a high molecular weight protein aggregate which is associated with free fatty acids (oleic, palmitoleic, linoleic) and is necessary for membrane activity. A variety of lipids will be further investigated in an attempt to determine their specificity. While reconstitution of excitability has been achieved in bilayers by using a variety of lipids, thus far the proteins used for unduction of excitability have all been of non-mammalian origin (EIM, alamethicin, monazomycin and the polyene DJ 400B). All of these display the same voltage dependent conductance as the excitable systems of nerve or muscle. In order to achieve complete reconstitution of action potential, we will attempt to use our new knowledge to develop methodologies for the isolation and characterization of the peptides or proteins responsible for excitability in mammalian nerve and muscle tissues. We will begin our work on bacterial products and then move up through hemocyanin and mitochondrial voltage dependent anionic channels (VDAC) to culminate our studies in mammalian systems. We will attempt to use our new knowledge to develop methodologies for the isolation and characterization of the peptides or proteins responsible for excitability in mammalian nerve and muscle tissues in order to achieve complete reconstitution of mammalian action potential.